The present invention relates generally to a method for determining the amount of hemoglobin in a biological sample such as feces, urine or gastric juice, and more particularly, to a quantitative method of determining the amount of such hemoglobin including converting the hemoglobin to porphyrin, isolating and separating out these porphyrins derived from hemoglobin or heme related compounds and determining the level of such porphyrins. This determination is preferably accomplished by means such as a fluorescence assay.
Various rapid screening tests for determining the presence of increased amounts of hemoglobin in biological materials such as feces, urine and gastric juice and currently available. These tests are used throughout the medical profession as a primary screening test for intestinal tumors. Some of these tests which are applicable primarily for feces are indirect tests based upon the peroxidase-like (pseudoperoxidase) activity of the hemoglobin. In these tests, colorless leuco dyes, in the presence of hemoglobin, become colored following addition of a suitable peroxide. These tests do not yield quantitative data and contain a relatively high percentage of false positive and false negative results. My prior patent application Ser. No. 190,399, filed Sept. 24, 1980, U.S. Pat. No. 4,378,971 describes an improved method for determining the level of hemoglobin in a biological sample such as feces, urine or gastric juice. This improved method enables the quantitative determination of hemoglobin in such sample by utilizing a concept of collecting and preparing the test sample, converting the non-fluorescing heme portion of the hemoglobin in the sample to fluorescing porphyrin and then assaying the fluorescence of the converted porphyrins. While this general approach and method are a significant improvement over the prior rapid screening non-quantitative techniques, and thus are highly satisfactory and acceptable in many cases, there is still room for further improvement in the accuracy of the quantitative determination.
One limitation of quantitative tests based upon a determination of converted porphyrins, such as the method described in my copending application referenced above, arises as a result of the existence of impurities in the test sample. These impurities alter the porphyrin determination, thus in turn affecting the accuracy of the hemoglobin determination. This is particularly true where the porphyrin determination is accomplished via a fluorescence assay. For example, biological materials such as feces contain many compounds which fluoresce at wavelengths similar to those of porphyrins derived from hemoglobin. These potential contaminants include naturally occurring porphyrins such as coproporphyrin which are not derived from hemoglobin and ingested prophyrins such as chlorophyll. These potential contaminants can also include numerous other compounds including various pigments and medicines whose fluorescence coincides with the region of maximum porphyrin fluorescence. The fluorescence of contaminants such as these normally exceeds that which is derived from the converted hemoglobin, and thus significantly affects the fluorescence assay in a test of the type described above.
Accordingly, there is a need for a procedure to purify and isolate the porphyrins derived from hemoglobin in a quantitative test for hemoglobin employing a porphyrin determination.